Caspase-1 is a member of a family of aspartate-specific cysteine proteases, known as the caspase family, involved in apoptosis and inflammation. Recent studies revealed that caspase-1 is activated within large protein complexes or assemblies called the inflammasomes. The inflammasomes are composed of several adaptor proteins that interact with caspase-1 and induce its oligomerization and activation. Preliminary evidence from the applicant's lab suggest that pyrin, a protein mutated in the auto- inflammatory disease Familial Mediterranean fever (FMF), plays a major role in inflammation by assembling an inflammasome complex with the adaptor protein ASC, and procaspase-1 leading to ASC oligomerization, caspase-1 activation and IL-1[unreadable] processing. Pyrin also appears to play an important role in apoptosis as macrophages from pyrin-truncation mice exhibit a defect in apoptosis compared to macrophages from wildtype mice in response to IL-4 and LPS stimulation. Evidence suggests that the inflammatory and perhaps the apoptotic activities of pyrin are regulated by two cytoskeleton associated proteins known as PSTPIP1 and PSTPIP2. Missense mutations in these proteins are associated with two further auto-inflammatory diseases, known as pyogenic arthritis, pyoderma gangrenosum, acne syndrome (PAPA) and chronic multifocal osteomyelitis (cmo) syndrome, respectively. In this competing continuation application three specific aims are proposed to study the physiological role of pyrin in apoptosis and inflammation and the role of the cytoskeleton in these two pyrin pathways. In the first specific aim, the role of pyrin in caspase-1 activation will be investigated through detailed biochemical and biological studies in cell-based and cell-free reconstitution systems and human and mouse macrophage cell lines. In the second specific aim, experiments are proposed to characterize the interaction of PSTPIP1 and PSTPIP2 with pyrin and determine their ability to regulate caspase-1 activation in human and mouse cells. In the third specific aim, biological, biochemical and genetic approaches will be used to elucidate the mechanism by which pyrin regulates apoptosis. Understanding the interplay of the pyrin-mediated inflammatory and apoptotic responses and the mechanism of activation of caspase-1 by pyrin is likely to contribute important insights into the role of pyrin in innate immunity and apoptosis, with obvious potential clinical applications. Public Health Relevance Statement: Mutations in the genes encoding pyrin and the pyrin-associated protein PSTPIP1 are associated with severe autoinflammatory diseases in humans. This research will investigate the physiological function of pyrin to elucidate its role in normal cellular function and how disease-associated mutations alter its function. The results of this research will help in the design and discovery of effective therapeutics to treat autoinflammatory diseases.